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The Journal of Experimental Biology 209, 000-000 Published by The Company of Biologists 2006 doi:10.1242/jeb.02274

Pharmacological characterization of the receptor in the salivary gland of the ixodid tick Amblyomma hebraeum W. Reuben Kaufman* and Jessi L. Minion Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9, Canada *Author for correspondence (e-mail: [email protected])

Accepted 18 April 2006

Summary Female ticks of the family Ixodidae osmoregulate by by 10·␮mol·l–1) and (22% maximum by secreting the excess fluid of the blood meal back into the 10·␮mol·l–1); and (D) one had no activity up to 1·mmol·l–1: host’s circulation via the salivary glands. At least three ergothioneine. Bromocriptine and did not receptors control salivary fluid secretion in the tick antagonize the action of DA, but were effective Amblyomma hebraeum: (1) (DA) stimulates fluid –1 competitive antagonists of ErN, with Kis of ~0.3·␮mol·l secretion via a DA receptor, (2) ergot (ErAs) and 11·␮mol·l–1, respectively. Ergothioneine was not an stimulate fluid secretion via an ErA-sensitive receptor (the antagonist of either the DA- or ErA-sensitive receptor. natural ligand of which has not been identified), and (3) a The putative protein kinase C activators, 1-oleoyl-2-acetyl- GABA receptor potentiates the action of DA and ErAs. sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DiC8), Here we present some pharmacological properties of the neither stimulated salivary fluid secretion nor potentiated ErA-sensitive receptor. Of the 11 ErAs we tested, (i) four the action of DA or ErN. The putative protein kinase C were complete (approximate concentration inhibitors, bisindolymaleimide (BIM) and calphostin C did eliciting 50% maximum response is given in parentheses): not inhibit the action of DA or ErN, although low ␮ –1 (0.02· mol·l ), ergonovine (ErN; concentrations of calphostin C (10·nmol·l–1) appeared to ␮ –1 ␮ –1 ␣ 0.06· mol·l ), methylergonovine (0.1· mol·l ) and - potentiate the action of DA but not ErN. The ion transport –1 ergocriptine (0.9·␮mol·l ); (ii) three were ‘incomplete inhibitors, furosemide and amiloride (both up to agonists’ (approximate concentration eliciting 20% 1·mmol·l–1), had no significant effect on DA-stimulated or maximum response is given in parentheses): ergocorninine ErN-stimulated fluid secretion. (3.5·␮mol·l–1), ergocristinine (7.5·␮mol·l–1) and ␮ –1 (10· mol·l ); (C) three were partial agonists Key words: Amblyomma hebraeum, bromocriptine, (approximate concentration eliciting the respective dihydroergotamine, , ergocorninine, ergocristine, maximum response in parentheses): ergocornine (50% ergocristinine, , ergonovine, ergothioneine, ergot maximum by 1·␮mol·l–1), methysergide (28% maximum alkaloids, methylergonovine, methysergide, tick salivary gland.

Introduction suggests that this receptor is of the GABAA type. The effect of Female ixodid ticks feed on a host for 7–14 days, during GABA is blocked by picrotoxin (100·␮mol·l–1) and (–)- which time they concentrate the nutrient part of the blood meal bicuculline (100·␮mol·l–1) in a non-competitive manner by secreting a large volume of hyposmotic saliva. The total (Lindsay and Kaufman, 1986). saliva secreted over the complete feeding period equals or The ergot alkaloid (ErA), ergonovine (ErN), is also a very exceeds the final engorged weight of the tick (reviewed by potent of salivary fluid secretion (Kaufman and Wong, Kaufman, 1989; Sauer et al., 2000; Bowman and Sauer, 2004). 1983). Results with selective antagonists demonstrate that ErN Control of fluid secretion is mediated via at least three acts via a receptor distinct from the DA receptor (Kaufman and receptors. Dopamine (DA) and other catecholamines stimulate Wong, 1983). We call this third receptor an ‘ErA-sensitive secretion via a DA receptor. This receptor appears to be in the receptor’ because we do not know the identity of its natural D-1 family (Schmidt et al., 1981; Schmidt et al., 1982). ␥- ligand. Aminobutyric acid (GABA) potentiates the fluid secretion Similar to other D-1 receptors, the effect of DA on this tissue stimulated by DA via a distinct receptor, but GABA has no is mediated by cAMP (Schmidt et al., 1981; Schmidt et al., intrinsic activity (Lindsay and Kaufman, 1986). The fact that 1982). Supramaximal concentrations of ErN also stimulate muscimol, but not baclofen, mimics the potentiation of GABA, adenylate cyclase activity in the tick salivary glands, but only 2 W. R. Kaufman and J. L. Minion to ~50% of the level stimulated by supramaximal University of Southampton, UK. The following drugs were concentrations of DA (W.R.K., unpublished observations), purchased from Sigma: DA-HCl, ergonovine-maleate, suggesting that the intracellular pathway mediating the action dihydroergotamine, ␣-ergocryptine, ergothioneine and 5- of ErN may be somewhat different from that of DA. hydroxytrypamine (5-HT). Structures of the ErAs used in this ErAs elicit an enormously broad spectrum of study [plus from Kaufman (Kaufman, 1977)] are pharmacological effects, the best known being at adrenergic, shown in Fig.·1. Deprenyl [monoamine oxidase (MAO) and tryptaminergic receptors (Berde and Schild, inhibitor] was purchased from Research Biochemicals Inc. 1978; Peroutka, 1996; Pertz and Eich, 1999), probably because (Natick, MA, USA). 1-oleoyl-2-acetyl-sn-glycerol (OAG), the structures of these biogenic amines can be exactly 1,2-dioctanoyl-sn-glycerol (DiC8), bisindolymaleimide (BIM) superimposed onto specific parts of the ring structure and calphostin C, were purchased from Calbiochem (San (Berde, 1980). The purpose of this study was to extend our Diego, CA, USA). characterization of the pharmacological properties of the ErA- Stock solutions of all drugs were made daily; DA, deprenyl, sensitive receptor in the tick salivary gland. We constructed ErN, methylergonovine, ergothioneine and methysergide were –1 dose–response curves for 11 ErAs available to us. Non- each dissolved in double-deionized water (to 1 or 10·mmol·l ) agonists and partial agonists were tested as putative inhibitors and then diluted to working concentration in modified TC 199. of the ErA and DA receptors. We also tested some inhibitors Calphostin C, OAG, DiC8, BIM, ergocryptine, bromocryptine, and activators of protein kinase C (PKC) for differential effects dihydroergotamine, ergocornine, ergocorninine, ergocristine, on the DA and ErN pathways. Likewise, we tested two ergocristinine were each dissolved in dimethylsulphoxide –1 –1 –1 –1 inhibitors of ion transport (amiloride and furosemide) for (DMSO; 0.5·mmol·l , 1·mmol·l , 2·mmol·l or 10·mmol·l ) differential effects on the two pathways. and then diluted to working concentration in modified TC 199 such that the final concentration of DMSO did not exceed 1%; we have demonstrated previously that this concentration of Materials and methods DMSO does not significantly affect the rate of salivary fluid Ticks secretion in vitro (Lindsay and Kaufman, 1986). All animal use in the Department of Biological Sciences, In vitro technique for measuring salivary fluid secretion University of Alberta, is reviewed by the Biosciences Animal The technique was similar to that described by Kaufman and Policy and Welfare Committee according to the Guidelines Phillips (Kaufman and Phillips, 1973b) and Wong and established by the Canadian Council on Animal Care. Kaufman (Wong and Kaufman, 1981). Glass Petri dishes Amblyomma hebraeum (Koch) were taken from a laboratory (15·cm diameter) were lined with paraffin wax into which four colony raised in darkness, at 26°C and high relative humidity. fine glass posts were embedded to serve as anchors for the Ticks were fed on rabbits as originally described for experimental glands. The Petri dishes were filled with liquid Dermacentor andersoni by Kaufman and Phillips (Kaufman paraffin (light mineral oil; Fisher Scientific, Town, State, and Phillips, 1973a). For this study we used partially fed ticks Country). Briefly, salivary glands were dissected out under in the range of 80–300·mg (normal engorged mass of A. modified Hank’s saline with their main ducts completely intact. hebraeum is in the range of 1000–3000·mg). The glands were transferred to modified TC 199 and extraneous tissue clinging to the main duct was teased away. Drugs and media Fine silk thread was ligated to a fragment of cuticle associated Modified Hank’s saline (dissection medium) with the terminal portion of the main duct, but without –1 Composition in g·l was: 11.5 NaCl, 1.6 D-glucose, 0.4 KCl, occluding the orifice. The glands were then transferred to the 0.14 CaCl2, 0.098 MgSO4, 0.06 KH2PO4, 0.05 NaHPO4, 0.01 Petri dish in a droplet of bathing medium such that the bathing Phenol Red. The pH was adjusted with NaOH to 7.2; the medium adhered to the glass post and the silk thread draped osmotic pressure was ~360·mOsm. over the far rim of the Petri dish. The thread was then gently pulled so that the main duct protruded from the bathing droplet Modified TC 199 (in-vitro bathing medium) into the mineral oil. Whenever the bathing medium contained 1 package powdered TC199 (11·g; Gibco, Town, State, an effective agonist, a small, spherical droplet of secreted fluid Country), 2.1·g NaCl and 2.09·g 3-[N- would appear and grow at the orifice of the main duct. For morpholino]propanesulfonic acid (Mops; Sigma Chemical measuring the amount of secreted fluid, the droplet was Co., St Louis, MO, USA) were dissolved in distilled-deionized removed from the orifice with a fine glass rod, allowed to sink, water, the pH was adjusted with NaOH to 7.2 and the final the diameter measured under a stereomicroscope fitted with an volume was brought to 1·l. ocular micrometer, and the sphere volume calculated from the The following drugs were a gift from Hoffman LaRoche diameter of the droplet. (Basle, Switzerland): bromocryptine, methylergonovine, ergocristine, ergocristinine, ergocornine, ergocorninine and General experimental protocol methysergide. (+/–)- (Ravizza, Italy or Delagrange, All experiments were done at room temperature France) was a gift from Dr G. N. Woodruff, formerly of the (22.5±0.5°C). Glands were exposed to a control treatment Ergot alkaloid receptor of Amblyomma hebraeum 3

(usually a given concentration of agonist), and the rate of changes of prevailing medium. Concentration of drug was secretion was recorded every 3–5·min until an equilibrated rate increased by 10-fold increments until the response was for that treatment was observed (usually 10–20·min). During maximal. When the drug under consideration was not DA, the this time, the prevailing medium was changed for a fresh glands were then washed in drug-free TC 199 for at least droplet every 3–5·min. Then the glands were exposed to 20·min, and then exposed to 10·␮mol·l–1 DA for three to five successively higher concentrations of drug, each time an 3-min readings. Unless otherwise stated, the response of the equilibrated rate of secretion being recorded following similar glands to 10·␮mol·l–1 DA was designated as 100%, and the

A Ergonovine (complete) B Methylergonovine (complete)

R1= –H, R2=–CH(C2H5)CH2OH C Ergotamine (complete; Kaufman, 1977) D Dihydroergotamine (complete)

E α-Ergocriptine (complete) F/G Ergocornine/inine (partial/incomplete)

H/I Ergocristine/inine (both incomplete) J Methysergide (partial)

R1= –H, R2=–CH(C2OH5)CH2OH5

K Bromocriptine (partial) L Ergothioneine (inactive)

Fig.·1. (A–L) Ergot alkaloids tested on tick salivary glands. The agonist activity for each is indicated in parentheses. All structures compiled from (Berde and Schild, 1978). Reproduced with kind permission of Springer Science and Business Media. 4 W. R. Kaufman and J. L. Minion maximal responses of the other treatments were compared to ergocryptine (0.9·␮mol·l–1). (2) Incomplete agonists were this value. those that neither elicited the maximum response nor appeared Putative antagonists were tested by one of two protocols. to reach some plateaued response at the highest concentration The first was a conventional dose–response protocol, in the that could be tested (usually because of limited availability). presence of the drug being tested for antagonism. In the second The approximate concentrations eliciting 20% maximum protocol, the glands were exposed to an agonist drug (usually response were: ergocorninine (3.5·␮mol·l–1), ergocristinine a sub-maximal concentration) until there was no further (7.5·␮mol·l–1), and ergocristine (10·␮mol·l–1; Fig.·3). (3) increase in secretory rate. Then putative antagonist was tested Partial agonists (Fig.·4) were those for which the response (several concentrations) until an equilibrated rate of secretion plateaued at substantially less than 100% of the maximum was observed at each concentration. To test for reversal of response. These were (with the approximate lowest antagonism, the glands were re-exposed to agonist alone for up concentration eliciting the respective maximum response in to 30·min. parentheses): ergocornine (50% maximum by 1·␮mol·l–1), –1 Inhibitory constants (Ki) were calculated from the following methysergide (28% maximum by 10·␮mol·l ) and equation: bromocryptine (22% maximum by 10·␮mol·l–1). (4) Non- agonists. Ergothioneine was the only ErA with no intrinsic K = (dose ratio – 1) / [I]·, i activity at the highest concentration tested (seven glands tested where dose ratio is the ratio of the two agonist concentrations up to 1·mmol·l–1). producing the same response in the presence and absence of As mentioned in the Introduction, ErAs interact frequently the inhibitor, I (Goldstein et al., 1974). with 5-HT and catacholamine receptors, behaving either as agonists or antagonists, but the natural ligand for the ErA- Statistics sensitive receptor in tick salivary glands has not yet been Unless otherwise stated, data are reported as mean ± s.e.m. identified. The effect of 5-HT on tick salivary glands has been (N), and statistically analysed using one-way ANOVA or tested in a number of studies, and at best it is a very weak Student’s t-tests. agonist (Kaufman and Phillips, 1973b; Kaufman, 1977; Needham and Sauer, 1975). We tested its action again as part of this study, with the hope of determining whether it acted via Results the DA receptor (blocked by ) or the ErA-sensitive Agonists receptor [blocked by sulpiride (Kaufman and Wong, 1983)]. The 11 ErAs tested in this study (Fig.·1) can be categorized Of 16 glands tested that responded to 10·␮mol·l–1 DA, only arbitrarily into four groups on the basis of the responses five were also stimulated by 5-HT (up to 10·mmol·l–1); 5-HT obtained. (1) Complete agonists (Fig.·2) were those that was an incomplete agonist, with 20% maximum response stimulated the maximum response at or below the highest occurring at ~220·␮mol·l–1 (Fig.·3). concentration tested. These were, in order of potency We asked whether this inconsistent and weak response to 5- (approximate concentration eliciting 50% maximum response HT might be due to high levels of monoamine oxidase (MAO) in parentheses): dihydroergotamine (0.02·␮mol·l–1), ErN in the tissue. So we repeated some dose–response experiments (0.06·␮mol·l–1), methylergonovine (0.1·␮mol·l–1), ␣- with 5-HT in the presence of 220·␮mol·l–1 deprenyl, an MAO

Methylergonovine (N=8) 50 Ergocristinine (N=4–8) 120 Dihydroergotamine (N=7) Ergocristine (N=7) Ergocryptine (N=7) 40 Ergocorninine (N=4) 100 5-HT (N=5) ErN (N=6) 80 30 60 20 40 % Maximum % Maximum 20 10

0 0 0.01 0.1 1 10 100 1000 10 000 0.001 0.01 0.1 1 10 100 Concentration (µmol l–1) –1 Concentration (µmol l ) Fig.·3. Incomplete agonists. Dose–response curves for the indicated Fig.·2. Complete agonists. Dose–response curves for the indicated drugs were conducted as described in Materials and methods. The drugs were constructed as described in Materials and methods. The s.e.m. is shown wherever it exceeds the size of the symbol. The data standard error of the mean (s.e.m.) is shown wherever it exceeds the for 5-hydroxytrypamine (5-HT) includes only those glands that size of the symbol. showed some response to 5-HT. Ergot alkaloid receptor of Amblyomma hebraeum 5

Bromocryptine (N=6) 120 A Methysergide (N=4) Control (N=4–13) Ergocornine (N=2–6) –1 60 100 100 µmol l bromocryptine (N=3) –1 50 80 1 µmol l methysergide (N=3) 40 60 30 40 20 % Maximum 20

% Maximum 10 0 0 0.001 0.01 0.1 1 10 DA concentration (µmol l–1) 0.01 0.1 1 10 100 –1 Concentration (µmol l ) Control (N=6) B 1 µmol l–1 bromocryptine (N=6) Fig.·4. Partial agonists. Dose–response curves for the indicated drugs 100 µmol l–1 bromocryptine (N=3) were conducted as described in Materials and methods. The s.e.m. is 120 shown wherever it exceeds the size of the symbol. 1 µmol l–1 methysergide (N=4) 100 80 inhibitor known to be effective on tick salivary glands (Kaufman and Sloley, 1996). Even though MAO should have 60 been almost completely inhibited under these conditions, 5-HT 40 % Maximum still failed to stimulate salivary fluid secretion in eight glands, 20 all of which responded to 10·␮mol·l–1 DA (data not shown). Thus we abandoned our attempt to determine at which receptor 0 5-HT elicits its weak, inconsistent effect. 0.001 0.01 0.1 1 10 100 Putative antagonists ErN concentration (µmol l–1) Ergothioneine (non-agonist), bromocryptine and methysergide (partial agonists) were tested for their ability to Fig.·6. Inhibition of (A) dopamine (DA) and (B) ergonovine (ErN) by inhibit ErN- and DA-stimulated secretion. Ergothioneine the partial agonists, bromocriptine and methysergide. The s.e.m. is caused no rightward shift of the ErN dose–response curve shown wherever it exceeds the size of the symbol. (Fig.·5) nor of the DA dose–response curve (five trials; data not shown). Bromocryptine (3 trials) and methysergide (3 trials) did not significantly antagonize the action of DA receptors by the differential antagonism of sulpiride (Kaufman (Fig.·6A), but both were effective, surmountable antagonists of and Wong, 1983). We confirmed these findings here, –1 –1 ErN (Fig.·6B), with Kis of ~0.3·␮mol·l (bromocryptine) and determining an inhibitory constant of ~0.73·␮mol·l for ~11·␮mol·l–1 (methysergide). sulpiride on ErN (Fig.·5), and an inability for sulpiride to DA and ErN have been demonstrated to act via distinct attenuate the action of DA (data not shown).

Control (N=6) Intracellular pathways 100 µmol l–1 sulpiride (N=8) The intracellular pathways mediating DA-induced secretion 500 µmol l–1 sulpiride (N=4) involve cAMP, Ca2+, cytosolic phospholipase A (cPLA ), and 120 2 2 Ergothioneine (N=4) arachidonic acid (reviewed by Sauer et al., 2000; Bowman and 100 Sauer, 2004). Nothing is known about the intracellular 80 pathways mediating ErA-induced secretion. However, there is 60 evidence that protein kinase C (PKC) is a component of the 40 intracellular signalling pathway in tick salivary glands (see

% Maximum 20 Discussion). So, in this study we tested the actions of a number 0 of drugs known to stimulate or inhibit PKC in other systems 0.001 0.01 0.1 1 10 100 1000 to learn whether the action of DA and ErN were affected differently. ErN concentration (µmol l–1) BIM (Toullec et al., 1991) and calphostin C (Kobayashi et Fig.·5. Inhibition of ergonovine (ErN) by putative antagonists, al., 1989; Bruns et al., 1991) were tested for possible inhibitory ergothioneine and (+/–)-sulpiride. The s.e.m. is shown wherever it effects, and OAG (Benz et al., 1992; Florin-Christensen et al., exceeds the size of the symbol. 1993) and DiC8 (Lapetina et al., 1985) were tested for possible 6 W. R. Kaufman and J. L. Minion excitatory effects on DA- and ErA-stimulated fluid secretion. Ion transport inhibitors Salivary glands were exposed to a sub-maximal concentration Furosemide [an inhibitor of various Cl– cotransporter of DA (0.1·␮mol·l–1) or ErN (0.1·␮mol·l–1) for six 5-min systems (Cabantchik and Greger, 1992)] and amiloride [an readings to determine the equilibrated response at that inhibitor of many Na+ transport systems (Kleyman and Cragoe, concentration; the mean value (± s.e.m.) of the last two or three Jr, 1988)] were both tested for effects on DA- and ErN- readings was recorded as the control response. Then the test stimulated secretion. Salivary glands were exposed to drug (PKC activator or inhibitor) was added in the presence of 10·␮mol·l–1 DA or 10·␮mol·l–1 ErN until maximum secretion DA or ErN and secretion was monitored for up to a further six was achieved. They were then exposed to DA or ErN along 5-min readings. The glands were then re-exposed to either DA with 1·␮mol·l–1 to 1·mmol·l–1 furosemide or amiloride for three or ErN alone to record recovery. The results for the putative successive readings of 5·min for each concentration. Neither PKC activators, OAG and DiC8, are presented in Table·1, and furosemide nor amiloride had any significant effect on DA- or those for the putative PKC inhibitors, BIM and calphostin C, ErN-stimulated secretory rates; note, however, that the sample are presented in Table·2 and Fig.·7. sizes were small (Table·3). Neither OAG nor DiC8 potentiated the action of sub- maximal concentrations of DA or ErN (Table·1). In all cases the secretory rates declined in the presence of OAG or DiC8 Discussion and on return to the control treatment, but at least part of this The multiple agonist and antagonist actions of ErAs in reduction may have been the natural decline that occurs with systems other than the tick salivary gland (most studies are on time when glands are continually stimulated for extensive mammals) are mediated predominantly, if not exclusively, via periods (Kaufman, 1976). various catecholamine and tryptamine receptors (Berde and BIM did not inhibit the secretory response of salivary glands Schild, 1978; Goldstein et al., 1980; Pertz and Eich, 1999). in the presence of sub-maximal (0.1·␮mol·l–1) or supra- Most studies on insects and other invertebrates conform to maximal (10·␮mol·l–1) concentrations of ErN or DA (Table·2) vertebrate models in this respect (e.g. Sakharov et al., 1989; The response to calphostin C depended on the concentration Wedemeyer et al., 1992; Blenau et al., 1998; Degen et al., range. Low concentrations of calphostin C (10·nmol·l–1) 2000). However, 5-HT is only a very weak agonist at best for appeared to potentiate the action of DA (leftward shift of the stimulating fluid secretion in tick salivary glands (Kaufman dose-response curve; Fig.·7A) but not that of ErN (Fig.·7B). At and Phillips, 1973b; Needham and Sauer, 1975; Kaufman, 0.1·␮mol·l–1 calphostin C, there was no statistically significant 1977) (this study). Evidence for a DA receptor (a D-1 type), inhibitory (or potentiating) effect using a sub-maximal however, is considerable (for reviews, see Sauer et al., 2000; (0.1·␮mol·l–1) concentration of ErN or DA (Table·2). Bowman and Sauer, 2004). As mentioned in the Introduction, ErAs do not exert their effects via this receptor. In brief, the ErA effects on the tick salivary gland cannot be interpreted in Table 1. Effect of the protein kinase C activators on the same light as ErA effects in most other systems. There dopamine- and ergonovine-stimulated fluid transport by tick seem to be only very few other examples in the literature in salivary glands which an ErA might act via an unidentified receptor (den Boer Treatment % Maximum* NP† et al., 1991). In this study, the ErAs tested fell into one of four classes, OAG grouped arbitrarily according to their efficacy at the ErA- DA (control) 85.1±3.8 8 – DA + OAG 79.9±4.4 8 0.382 sensitive receptor: complete agonists, incomplete agonists, Return to DA 63.8±4.4 8 0.002 partial agonists and non-agonists. The structures of the ErAs ErN (control) 87.5±2.2 7 – tested are shown in Fig·1. Those that exhibited at least some ErN + OAG 72.4±9.9 7 0.183 agonist activity (all except ergothioneine) are based on an Return to ErN 55.4±10.8 7 0.025 ergoline ring structure. Although ergotamine was not tested DiC8 here, it has also been reported as a full agonist, with a potency DA (control) 78.6±3.5 8 – similar to that of ergonovine (Kaufman, 1977). There is little DA + DiC8 50.6±6.0 10 0.001 beyond that broad generalization, however, to explain the other Return to DA 43.4±6.5 4 0.005 differences in activity presented here. For example, only three ErN (control) 78.6±4.8 9 – of the five complete agonists are ergopeptines (ergotamine, ErN + DiC 59.2±3.0 9 0.004 8 dihydroergotamine and ␣-ergocriptine), the other two (ErN and Return to ErN 41.6±2.9 9 0.0000 methylergonovine) are not. Moreover, with the exception of –1 methysergide, all of the incomplete and partial agonists are OAG, 1-oleolyl-2-acetyl-sn-glycerol (100·␮mol·l ); DiC8,1,2- dioctanoyl-sn-glycerol (100·␮mol·l–1); DA, dopamine (0.1·␮mol·l–1); also ergopeptines (ergocornine, ergocorninine, ergocristine, ErN, ergonovine (0.1·␮mol·l–1). ergocristinine and bromocriptine). *% Maximum is calculated from the maximum rate recorded It is commonly observed that the action of ErAs is difficult during each initial control period (± s.e.m.). to reverse even with extensive washout procedures (Bond et †Student’s t-test (cf control). al., 1989; Schöning et al., 2001), and this was found to be the Ergot alkaloid receptor of Amblyomma hebraeum 7

Table 2. Effect of the protein kinase C inhibitors, bisindolymaleimide and calphostin C on ergonovine- and dopamine-stimulated fluid transport by tick salivary glands Treatment % Maximum* NP† BIM 10·␮mol·l–1 ErN (control) 82.3±3.1 5 – 10·␮mol·l–1 ErN + 0.1·␮mol·l–1 BIM 79.9±3.0 5 0.589 10·␮mol·l–1 ErN + 0.3·␮mol·l–1 BIM 93.5±10.4 5 0.343 10·␮mol·l–1 ErN + 1.0·␮mol·l–1 BIM 79.3±11.2 4 0.810 0.1·␮mol·l–1 ErN (control) 82.7± 2.0 4 – 0.1·␮mol·l–1 ErN + 0.2·␮mol·l–1 BIM 105±14.6 4 0.227 Return to 0.1·␮mol·l–1 ErN 66.0±3.2 3 0.015 0.1·␮mol·l–1 DA (control) 87.2±4.8 4 – 0.1·␮mol·l–1 DA + 0.2·␮mol·l–1 BIM 97.1±14.4 4 0.554 Return to 0.1·␮mol·l–1 DA 108±7.8 3 0.095 Calphostin C 0.1·␮mol·l–1 ErN (control) 83.9±2.9 5 – 0.1·␮mol·l–1 ErN + 0.1·␮mol·l–1 calphostin C 90.5±3.6 5 0.194 Return to 0.1·␮mol·l–1 ErN 48.9±0.5 2 0.0002 0.1·␮mol·l–1 DA (control) 82.7±4.1 6 – 0.1·␮mol·l–1 DA + 0.1·␮mol·l–1 calphostin C 106±10.1 6 0.077 Return to 0.1·␮mol·l–1 DA 104±17.7 5 0.398

BIM, bisindolymaleimide; DA, dopamine; ErN, ergonovine. *% Maximum is calculated from the maximum rate recorded during each initial control period (± s.e.m.). †Student’s t-test (cf control). case with the tick salivary gland. One potential explanation for Berridge and Prince (Berridge and Prince, 1973), reporting on the long-lasting effect is continual diffusion from a pool of the agonist action of LSD at the blowfly salivary gland 5-HT drug absorbed into the tissue during the incubation period receptor, discounted the latter possibility for that system. They (Bulow et al., 1986; Tfelt-Hansen and Johnson, 1993). proposed instead that part of the ErA molecule (presumably the diethyl amide) binds tightly to an allosteric site of the 5- HT receptor, allowing the active portion (presumably the 120 A 100 ergoline) to repeatedly attach to and disengage from the active 80 site. Similar to the blowfly salivary gland, the tick salivary gland is free of layers of muscle or connective tissue that could 60 serve to store a pool of drug (although extrinsic fat body tissue 40 is omnipresent). In rat tail artery preparations, insurmountable % Maximum 20 Control (N=4–13) blockade of the 5-HT response by methysergide has been 0 –1 10 nmol l calphostin C (N=3) attributed to allosteric modulation of 5-HT2A receptors rather than to pseudoirreversible inhibition (Pertz and Eich, 1992). In 0.001 0.01 0.1 1 10 100 this case, however, (unlike that for the blowfly and tick salivary –1 DA concentration (µmol l ) glands), onset of action is also very prolonged (Schöning et al., 2001). A general model has been proposed whereby one can 120 B differentiate between slow ligand–receptor dissociation from 100 slow diffusion (Martin et al., 1995). 80 The results presented here further support earlier 60 observations that 5-HT is unlikely to be the endogenous ligand 40 at the ErA-sensitive receptor. The small and inconsistent effect of 5-HT might be due to an indirect action. For example, tick % Maximum 20 Control (N=6) salivary glands contain a relatively high endogenous content 10 nmol l–1 calphostin C (N=3) 0 of DA (Kaufman and Sloley, 1996), possibly in the numerous granular cells. Perhaps the inconsistent agonist action of 5-HT 0.001 0.01 0.1 1 10 100 is due to an occasional stimulation of DA release from this –1 ErN concentration (µmol l ) endogenous source. Fig.·7. Potentiation of (A) dopamine (DA) but not (B) ergonovine There is now a growing understanding about the intracellular (ErN) by the putative PKC inhibitor calphostin C. The s.e.m. is shown signalling cascade following stimulation of the tick wherever it exceeds the size of the symbol. (Amblyomma americanum) salivary gland DA receptor 8 W. R. Kaufman and J. L. Minion

Table 3. Effect of amiloride and furosemide on dopamine- and ergonovine-stimulated fluid transport by tick salivary glands Treatment % Maximum* NP† Amiloride DA (control) 100±0 6 – DA + 1·␮mol·l–1 amiloride 108±6.9 3 0.369 DA + 10·␮mol·l–1 amiloride 104±15.0 3 0.832 DA + 100·␮mol·l–1 amiloride 99.8±11.1 6 0.986 DA + 1·mmol·l–1 amiloride 98.4±13.6 2 0.925 DA + 10·mmol·l–1 amiloride 83.7±28.3 2 0.667 ErN (control) 100±0 2 – ErN + 1·␮mol·l–1 amiloride 112±22.6 2 0.701 ErN + 10·␮mol·l–1 amiloride 112±12.4 2 0.500 ErN + 100·␮mol·l–1 amiloride 94.3±15.3 2 0.642 Furosemide DA (control) 100±0 2 – DA + 1·␮mol·l–1 furosemide 123±11.8 2 0.218 DA + 10·␮mol·l–1 furosemide 134±0 2 – DA + 100·␮mol·l–1 furosemide 122±0 2 – DA + 1·mmol·l–1 furosemide 131±3.1 2 0.063 ErN (control) 100±0 3 – ErN + 1·␮mol·l–1 furosemide 104±2.7 3 0.190 ErN + 10·␮mol·l–1 furosemide 102±2.3 3 0.765 ErN + 100·␮mol·l–1 furosemide 96.8±9.4 3 0.684

DA, dopamine (10·␮mol·l–1); ErN, ergonovine (10·␮mol·l–1). *% Maximum is calculated from the maximum rate recorded during each initial control period (± s.e.m.). †Student’s t-test (cf control).

(reviewed by Sauer et al., 2000; Bowman and Sauer, 2004). trivolvis (Christopher et al., 1999), inhibition of PKC by BIM in The salivary gland contains several isoforms of a cAMP- chick proximal tubule (Dudas et al., 2002), activation of PKC in dependent protein kinase (cAPKC1-3) that are activated in rabbit portal vein myocytes (Albert and Large, 2001), isolated salivary glands exposed to DA. As a result, at least a stimulation of PKC of human kidney proximal tubules (Pietig et dozen salivary gland proteins are phosphorylated. The glands al., 2001), and inhibition of PKC by calphostin C on human also contain phosphoprotein phosphatase activity, further endothelial cells (Orzechowski et al., 2001). supporting a role for PKC in salivary fluid secretion. Under the Our knowledge of the pharmacological properties of the tick conditions tested here, there is little evidence to suggest that ErA-sensitive receptor is obviously rudimentary compared to PKC is an intermediate in the pathway linked to the ErA- that of many mammalian systems, at least in part because sensitive receptor or the DA receptor. The two PKC activators, relatively few congeners have been tested. Recently it has been OAG and DiC8, did not stimulate salivary fluid secretion on shown that 8R- is a partial agonist at human histamine their own (data not shown), and they did not potentiate the H1 receptors (Bakker et al., 2004), which expands the stimulatory effect of sub-maximal concentrations of DA or repertoire of ErA targets yet further. However, histamine (up ErN (both at 0.1·␮mol·l–1; Table·1). The PKC inhibitors, BIM to 1·mmol·l–1) did not stimulate fluid secretion in tick salivary and calphostin C, likewise did not inhibit the stimulatory action glands (Kaufman, 1977). Unfortunately, we still have no idea of 0.1·␮mol·l–1 DA or ErN (Table·2); if anything, the trend was as to what the natural ligand is at the tick salivary gland ErA- to potentiate the action of DA and ErN, though the differences sensitive receptor, or whether it is a component of a neuronal between control and treated samples in Table·2 were not or hormonal pathway. We also do not know the physiological statistically significant in a consistent manner. The potentiating conditions under which this pathway to fluid secretion is trend was also observed at lower concentrations of calphostin stimulated [the DA pathway seems to be involved in C, where 10·nmol·l–1 appeared to shift the dose–response curve haemolymph volume regulation (Kaufman et al., 1980)]. This of DA to the left, but not that of ErN (Fig.·7); the number of remains a major gap in our understanding of tick salivary gland replicates in this experiment, however, was small. This matter function. clearly merits further investigation. We have some confidence that the experimental protocol used for the PKC experiments was appropriate, both with regard to List of abbreviations the concentration ranges tested and the duration of incubation. 5-HT 5-hydroxytrypamine Just a few examples include: PKC mediation in 5-HT stimulated BIM bisindolymaleimide ciliary beat frequency in embryos of the pond snail, Heliosoma cPLA2 cytosolic phospholipase A2 Ergot alkaloid receptor of Amblyomma hebraeum 9

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